Bis-azo compounds with 2,3-dichlorobenzoyl group

ABSTRACT

A bis-azo compound represented by the following general formula (1), a tautomer thereof or a salt thereof: ##STR1## wherein R represents a substituted or unsubstituted phenyl group or a substituted or unsubstituted heteroaryl group and X represents a substituted or unsubstituted phenylene group. These compounds exhibit low toxicity and do not have any undesirable effect such as mutagenecity.

TECHNICAL FIELD

The present invention relates to a novel bis-azo compound.

BACKGROUND ART

It has been known that azo compounds have wide variety of uses in, forinstance, dyes, dyestuffs for printing, food colors, liquid crystals anddrugs. Among these, bis-azo compounds each having two azo bonds in themolecule are compounds which have generally been known as dyes, inparticular, photographic light-sensitive materials used in the silverdye bleach process (see, for instance, U.S. Pat. Nos. 3,754,923 and3,671,253). Bis-azo compounds used as photographic light-sensitivematerials are characterized in that they have, in combination,hydrophilic groups (more specifically, a plurality of, for instance,sulfonate residues) for imparting moderate solubility in water to themolecules and hydrophobic groups and a molecular weight required forrendering the molecules uniformly dispersible in emulsions.

Such compounds should have toxicity as low as possible and should nothave undesirable properties such as mutagenicity. However, the studiesof the inventors of this invention and recent studies (MutationResearch, 1992, 277, p. 201) make it clear that most of theconventionally known bis-azo compounds show mutagenicity. For thisreason, there has been desired for the development of compounds whichare much safer and more useful in this regard.

DISCLOSURE OF THE INVENTION

Accordingly, an object of the present invention is to provide a novelbis-azo compound which is free of any undesirable effect such asmutagenicity.

The inventors of this invention have conducted various studies toachieve the foregoing object and as a result, have found out thatbis-azo compounds represented by the following general formula (1) or(2) whose acyl group attached to an amino group on anaminonaphthalenesulfonate moiety present in the compounds is modifiedhave low toxicity and are free of such undesirable effects asmutagenicity and thus have completed the present invention. ##STR2##

In Formula (1), R represents a substituted or unsubstituted phenyl groupor a substituted or unsubstituted heteroaryl group and X represents asubstituted or unsubstituted phenylene group. In Formula (2), Rrepresents a substituted or unsubstituted phenyl group or a substitutedor unsubstituted heteroaryl group and A and B may be the same ordifferent and each represents a group selected from the group consistingof hydrogen atom, alkyl groups having 1 to 4 carbon atoms, alkoxy groupshaving 1 to 4 carbon atoms and halogen atoms.

The present invention relates to a bis-azo compound which ischaracterized in that the N-acyl group present on8-amino-1-hydroxynaphthalene-3,6-disulfonic acid is 2,3-dichlorobenzoylgroup. Bis-azo compounds whose N-acyl group is p-chlorobenzoyl,m-chlorobenzoyl, 2,4-dichlorobenzoyl or 3,4-dichlorobenzoyl group aredisclosed in the U.S. Patents listed above, but most of these compoundsshow mutagenicity.

Contrary to this, the bis-azo compound substituted with a2,3-dichlorobenzoyl group and represented by the foregoing generalformula (1) or (2) has substantially reduced mutagenicity.

If R represents a substituted phenyl group, examples of preferredsubstituents thereof are halogen atoms, trifluoromethyl group, alkylgroups, alkoxy groups, alkylcarbonyl groups, aryl groups, aryloxygroups, arylcarbonyl groups, cyano group and hydroxyl group, withhalogen atoms such as fluorine or chlorine atom, trifluoromethyl group,cyano group, alkyl groups having 1 to 4 carbon atoms or alkoxy groupshaving 1 to 4 carbon atoms being more preferred as such a substituent.Among these, particularly preferred are halogen atoms, in particular,chlorine atom. Moreover, an unsubstituted phenyl group is likewisepreferred as the substituent R.

If R represents a chlorine atom-substituted phenyl group, the preferrednumber of chlorine atoms present on the phenyl group ranges from 1 to 3and preferably 1 or 2. If R is a monochloro-substituted phenyl group,the chlorine atom may be on either of o-, m- or p-position, inparticular, o-position. If R is a dichloro-substituted phenyl group,preferred examples thereof are 2,4-dichlorophenyl, 3,4-dichlorophenyl,2,3-dichlorophenyl and 2,5-dichlorophenyl groups, with2,4-dichlorophenyl, 2,5-dichlorophenyl and 2,3-dichlorophenyl being morepreferred and 2,4-dichlorophenyl being particularly preferred.

If the substituent R is a heteroaryl group, specific examples ofheteroaryl groups include pyridyl group, thienyl group, furyl group,quinolyl group and isoquinolyl group. If the heteroaryl group has asubstituent, examples of such substituents suitably include halogenatoms such as fluorine, chlorine, bromine and iodine atoms,trifluoromethyl group, alkyl groups, alkoxy groups, aryl groups, cyanogroup and hydroxyl group, with halogen atoms such as fluorine andchlorine and alkyl groups having 1 to 4 carbon atoms being preferred.

If the substituent R represents a substituted or unsubstitutedheteroaryl group, specific examples of R include 3-pyridyl, 4-pyridyl,2-thienyl, 2-furyl and 3-quinolyl groups, with 3-pyridyl and 3-quinolylgroups being particularly preferred.

If X represents a substituted phenylene group, the substituents may bethose listed above, as preferred substituents, in connection to the casewherein the substituent R is a phenyl group.

In the general formula (2), A and B may be the same or different andeach represents a substituent selected from the group consisting ofhydrogen atom, alkyl groups having 1 to 4 carbon atoms, alkoxy groupshaving 1 to 4 carbon atoms and halogen atoms. Preferred examples ofalkyl groups are methyl, ethyl and butyl groups; preferred examples ofalkoxy groups are methoxy, ethoxy and butoxy groups; and preferredhalogen atoms are fluorine, chlorine and bromine atoms. Among these,preferred are alkyl groups and alkoxy groups, in particular, alkoxygroups.

A and B may be the same or different, but preferably they represent thesame groups. Preferably, both A and B are alkoxy groups each having 1 to4 carbon atoms and most preferably, both A and B are methoxy groups.

Preferred combinations of R, A and B in the compounds represented by theforegoing general formula (2) are listed in Table 1. Among thesecombinations, preferred are (a), (b), (c), (d), (e), (f), (g), (h), (i),(j), (k) and (o) and particularly preferred combinations thereof are(c), (d), (e), (f), (g), (h), (i), (j) and (k).

                  TABLE 1                                                         ______________________________________                                        Combina-                                                                             Substituents                                                           tion   R               A          B                                           ______________________________________                                        (a)    phenyl group    methyl group                                                                             methyl group                                (b)    phenyl group    methoxy group                                                                            methoxy group                               (c)    monochlorophenyl group                                                                        methyl group                                                                             methyl group                                (d)    monochlorophenyl group                                                                        methoxy group                                                                            methoxy group                               (e)    monochlorophenyl group                                                                        ethoxy group                                                                             ethoxy group                                (f)    dichlorophenyl group                                                                          methyl group                                                                             methyl group                                (g)    dichlorophenyl group                                                                          methoxy group                                                                            methoxy group                               (h)    dichlorophenyl qroup                                                                          ethoxy group                                                                             ethoxy group                                (i)    dichlorophenyl group                                                                          hydrogen atom                                                                            hydrogen atom                               (j)    dichlorophenyl group                                                                          halogen atom                                                                             hydrogen atom                               (k)    dichlorophenyl group                                                                          methoxy group                                                                            methyl group                                (l)    3-pyridyl group methyl group                                                                             methyl group                                (m)    3-pyridyl group methoxy group                                                                            methoxy group                               (n)    3-quinolyl group                                                                              methyl group                                                                             methyl group                                (o)    3-quinolyl group                                                                              methoxy qroup                                                                            methoxy group                               ______________________________________                                    

The ionic group present in the bis-azo compound of the present inventionmay form a salt with an appropriate counterion, specifically a cation.The cation is preferably one which is substantially non-toxic and doesnot independently have significant pharmacological activity. Specificexamples of such salts are alkali metal salts such as sodium andpotassium salts, alkaline earth metal salts represented by magnesiumsalt, salts with light metals of Group IIIA including aluminum, ammoniumsalts, and salts with organic primary, secondary and tertiary amines.Among these salts, particularly preferred are sodium, potassium,ammonium salts and organic amine salts. Preferred examples of organicamines are triethylamine, tris(hydroxymethyl)aminomethane, andderivatives of amino acids and oligopeptides.

Alternatively, the bis-azo compounds of the present invention may alsobe used in the form of pharmaceutically unacceptable salts depending onthe applications thereof. Examples of such salts include thosecontaining barium, titanium, and zinc.

The method for synthesizing the bis-azo compounds of the presentinvention will now be described below. The compounds represented by theforegoing general formula (2) can easily be prepared from known startingmaterials or intermediates according to methods almost similar to thosedisclosed in U.S. Pat. Nos. 3,754,923 and 3,671,253 or those disclosedin published volumes for instance, Yutaka HOSODA, "Riron Seizo SenryoKagaku (Theoretical Manufacture and Chemistry of Dyes)", GihodoPublishing Company!.

For instance, the compounds represented by the foregoing general formula(2) can be prepared by first coupling a compound represented by thefollowing intermediate I with a diazonium salt prepared from a compoundrepresented by the following general formula (3) (in Formula (3), A andB are the same as those defined above) to form a monoazo compoundrepresented by the following general formula (4) (in Formula (4), A andB are the same as those defined above), then converting the monoazocompound into an amine derivative thereof represented by the followinggeneral formula (5) (wherein A and B are the same as those definedabove) through reduction of the nitro group present in the compound,finally coupling a diazonium salt prepared from the amine derivativewith a compound represented by the following general formula (6)(wherein the formula R is the same as that defined above), andoptionally salting out the resulting reaction system and filtering offthe precipitations thus formed. ##STR3##

In the series of steps described above, the first coupling step and thereducing step may be carried out in succession without isolating thenitro derivative represented by Formula (4). There is no doubt that thecompounds represented by Formula (2) may likewise be prepared bychanging the order of the coupling reactions, i.e., by first couplingthe diazonium salt prepared from the compound represented by Formula (3)with the compound represented by Formula (6), then reducing the nitrogroup present in the resulting product, converting it into a diazoniumsalt, and coupling the diazonium salt with the intermediate 1. In theforegoing description of the production method, the sulfonate residue isexpressed in terms of its free acid form, but the compounds whosesulfonate residues are in the form of salts with appropriate counterionsmay be used in the foregoing reaction to thus isolate intended bis-azocompounds. In this case, it is preferred that these compounds arereacted in the form of sodium salts and then intended bis-azo compoundsare isolated, because of easy availability of starting materials andreagents to be used. In addition, if it is needed to prepare a salt ofthe desired bis-azo compound with a counterion other than sodium ion, itcan be prepared by ion-exchanging the corresponding sodium salt.

Specific examples of the bis-azo compounds of the present invention willbe listed below (expressed in terms of sodium salts except for Compounds11 and 12), but the present invention is not restricted to thesespecific examples. ##STR4##

The present invention will hereinafter be described in more detail withreference to the following Examples,

EXAMPLE 1 Synthesis of Compound 1

The method for synthesizing Compound 1 will be detailed below. The routeof synthesis of Compound 1 is as follows: ##STR5## 1) Synthesis ofIntermediate 1

The intermediate was prepared by an improved Schotten-Baumann method.More specifically, 8-amino-3,6-disulfo-1-naphthol (H-acid, 34.2 g; 100mmol as expressed in terms of monosodium salt), sodium hydroxide (5.0 g,125 mmol) and sodium carbonate (37.0 g, 350 mmol) were dissolved indeionized water (200 ml) and a solution of 2,3-dichlorobenzoyl chloride(23.1 g, 110 mmol) in THF (20 ml) was dropwise added to the resultingsolution at 35° to 40° C. for about one hour while blowing nitrogen gasthrough the solution. The reaction mixture was vigorously stirred atthat temperature for one hour, then the temperature of the mixture wasraised up to 80° C. and the mixture was again vigorously stirred for onehour. After addition of 250 ml of a 10% aqueous common salt solution tothe mixture and allowing it to cool down to room temperature, theprecipitates formed were collected through filtration, followed bywashing the precipitates with a 10% aqueous common salt solution andacetonitrile in this order and then drying to give 40.0 g of the desiredintermediate 1 (yield 75% as calculated on the basis of the disodiumsalt thereof).

2) Synthesis of Intermediate 2

There was dissolved 2,5-dimethoxy-4-nitroaniline (4 g, 20 mmol) in amixed solution comprising deionized water (20 ml) and concentratedhydrochloric acid (5.1 ml) and then a solution of sodium nitrite (1.56g, 22 mmol) in deionized water (10 ml) was added to the resultingsolution while ice-cooling the latter. The reaction mixture was stirredfor 60 minutes with ice-cooling. The aqueous solution of the diazoniumsalt thus prepared was added to a solution (200 ml) of Intermediate 1(12 g, 22.2 mmol as calculated on the basis of its disodium salt form)and sodium acetate (5.5 g) in deionized water at 10° C. After stirringthe reaction mixture at 20° C. for one hour, it was heated to 45° C. andfurther stirred for additional one hour.

To the reaction mixture, therewere, in order, added a 20% aqueoussolution of sodium hydroxide (9 ml) and then sodium sulfide nonahydrate(19.2 g, 80 mmol), followed by stirring at 45° C. for one hour. Afterthe completion of the stirring operation, isopropyl alcohol (50 ml) wasadded to the reaction mixture and then the reaction mixture wasneutralized by the addition of acetic acid (9 ml) to thus giveprecipitates. Further a saturated sodium acetate aqueous solution (30ml) was added to the reaction system, followed by recovering theprecipitates thus formed through filtration and washing the precipitateswith a mixed solvent of a 10% aqueous sodium acetate solution andisopropyl alcohol (volume ratio 1:1) and then isopropyl alcohol.

The crude product of Intermediate 2 thus prepared was suspended in amixed solvent comprising toluene (160 ml) and isopropyl alcohol (40 ml)and then vigorously stirred under reflux. After the stirring operationunder reflux, the precipitates were collected through filtration,followed by washing with a mixed solvent of toluene and isopropylalcohol (volume ratio 4:1) and drying to give 12.2 g of the desiredIntermediate 2 (yield 84%, as calculated on the basis of the disodiumsalt thereof).

3) Synthesis of Compound 1

A solution of Intermediate 2 (5 g, 7.0 mmol) in deionized water (70 ml)was ice-cooled, followed by addition of concentrated hydrochloric acid(1.75 ml), vigorous stirring, addition of a solution of sodium nitrite(588 mg, 8.4 mmol) in deionized water (5 ml) and stirring the mixturefor 60 minutes with ice-cooling to give a diazonium salt. On the otherhand, Intermediate 1 (4.5 g, 8.4 mmol) was dissolved in deionized water(30 ml), followed by addition of pyridine (15 ml) and then addition of asuspension of the diazonium salt prepared by the foregoing procedures ata temperature ranging from 10° to 15° C. The reaction mixture wasstirred at room temperature for 60 minutes, then heated up to 50° C. andagain stirred for additional 30 minutes. Then the reaction mixture washeated to 70° C., followed by addition of isopropyl alcohol (100 ml) anda saturated aqueous solution of sodium acetate (40 ml). After coolingdown to 40° C., the precipitates thus formed were collected throughfiltration, followed by washing with a 10% aqueous sodium acetatesolution, a mixed solvent of isopropyl alcohol and water (volume ratio4:1) and isopropyl alcohol in this order and drying them.

The crude product of Compound 1 thus prepared was dissolved in 80 ml ofwater at 80° C. and then isopropyl alcohol (320 ml) was dropwise addedto the resulting solution at 70° C. with stirring. The mixture wascooled to 50° C., followed by collection of the resulting precipitatesthrough filtration, washing them with a mixed solvent of isopropylalcohol and water (volume ratio 4:1) and isopropyl alcohol in this orderand drying the precipitates to give 4.95 g (3.92 mmol, 56%) of Compound1.

EXAMPLE 2 Synthesis of Compound 2

The method for synthesizing Compound 2 will be detailed below. The routeof synthesis of Compound 2 is as follows: ##STR6## 1) Synthesis ofIntermediate 3

The intermediate was prepared by an improved Schotten-Baumann method.More specifically, 8-amino-3,6-disulfo-1-naphthol (H-acid, 68.2 g; 200mmol as calculated on the basis of its monosodium salt), sodiumhydroxide (8.6 g, 140 mmol) and sodium carbonate (12.7 g, 120 mmol) weredissolved in deionized water (400 ml) and 2,4-dichlorobenzoyl chloride(46.1 g, 220 mmol) was dropwise added to the resulting solution at 38°to 44° C. for about one hour while blowing nitrogen gas through thesolution. The reaction mixture was vigorously stirred at thattemperature for one hour, then the temperature of the mixture was raisedup to 80° C. and the mixture was again vigorously stirred for one hour.After addition of 80 ml of a 10% aqueous common salt solution to themixture and allowing it to cool down to 35° C., the precipitates formedwere collected through filtration, followed by washing the precipitateswith a 10% aqueous common salt solution and acetonitrile in this orderand then drying to give 82 g of the desired intermediate 3 (yield 77% ascalculated on the basis of its disodium salt).

2 ) Synthesis of Compound 2

A solution of Intermediate 2 (10 g, 14 mmol) in deionized water (120 ml)was ice-cooled, followed by addition of concentrated hydrochloric acid(3.5 ml), vigorous stirring, addition of a solution of sodium nitrite(1.18 g, 17 mmol) in deionized water (10 ml) and stirring the mixturefor 60 minutes with ice-cooling to give a diazonium salt. On the otherhand, Intermediate 3 (9 g, 16.8 mmol) was dissolved in deionized water(60 ml), followed by addition of pyridine (30 ml) and then addition of asuspension of the diazonium salt prepared by the foregoing procedures ata temperature ranging from 10° to 15° C. The reaction mixture wasstirred at room temperature for 60 minutes, then heated up to 50° C. andagain stirred for additional 30 minutes. Then the reaction mixture washeated to 70° C., followed by addition of isopropyl alcohol (200 ml) anda saturated aqueous solution of sodium acetate (60 ml). After coolingdown to 50° C., the precipitates thus formed were collected throughfiltration, followed by washing with a 10% aqueous sodium acetatesolution, a mixed solvent of isopropyl alcohol and water (volume ratio4:1) and isopropyl alcohol in this order and drying them.

The crude product of Compound 2 thus prepared was dissolved in 150 ml ofwater at 80° C. and then isopropyl alcohol (600 ml) was dropwise addedto the resulting solution at 70° C. with stirring. The mixture wascooled to 50° C., followed by collection of the resulting precipitatesthrough filtration, washing them with a mixed solvent of isopropylalcohol and water (volume ratio 4:1) and isopropyl alcohol in this orderand drying to give 9.5 g (7.5 mmol, 54%) of Compound 2.

EXAMPLE 3 Synthesis of Compound 4

1) Synthesis of Intermediate 4

The following Intermediate 4 was quantitatively prepared by repeatingthe same procedures used in the synthesis of Intermediate 1 in Example 1except that 3,4-dichlorobenzoyl chloride was substituted for2,3-dichlorobenzoyl chloride: ##STR7## 2) Synthesis of Compound 4

A solution of Intermediate 2 (3 g, 4.2 mmol) in deionized water (40 ml)was ice-cooled, followed by addition of concentrated hydrochloric acid(1.05 ml), vigorous stirring, addition of a solution of sodium nitrite(350 mg, 5.1 mmol) in deionized water (5 ml) and stirring the mixturefor 60 minutes with ice-cooling to give a diazonium salt. On the otherhand, Intermediate 4 (2.7 g, 5 mmol) was dissolved in deionized water(20 ml), followed by addition of pyridine (15 ml) and then addition of asuspension of the diazonium salt prepared by the foregoing procedures ata temperature ranging from 10° to 15° C. The reaction mixture wasstirred at room temperature for 60 minutes, then heated up to 50° C. andagain stirred for additional 30 minutes. Then the reaction mixture washeated to 70° C., followed by addition of isopropyl alcohol (100 ml) andsodium acetate (6 g). After cooling down to 40° C., the precipitatesthus formed were collected through filtration, followed by washing witha 10% aqueous sodium acetate solution, a mixed solvent of isopropylalcohol and water (volume ratio 4:1) and isopropyl alcohol in this orderand drying them.

The crude product of Compound 4 thus prepared was dissolved in 130 ml ofwater at 80° C. and then isopropyl alcohol (300 ml) was dropwise addedto the resulting solution at 70° C. with stirring. The mixture wascooled to 50° C., followed by collection of the resulting precipitatesthrough filtration, washing them with a mixed solvent of isopropylalcohol and water (volume ratio 4:1) and isopropyl alcohol in this orderand drying to give 2.7 g (2.14 mmol, yield 51%) of Compound 4.

EXAMPLE 4 Synthesis of Compound 5

1) Synthesis of Intermediate 5 ##STR8##

The intermediate was prepared by an improved Schotten-Baumann method.More specifically, 8-amino-3,6-disulfo-1-naphthol (H-acid, 51.2 g; 150mmol as calculated on the basis of its monosodium salt), sodiumhydroxide (7.5 g, 188 mmol) and sodium carbonate (10.6 g, 120 mmol) weredissolved in deionized water (300 ml) and o-chlorobenzoyl chloride (29g, 165 mmol) was dropwise added to the resulting solution at 35° to 40°C. for about one hour while blowing nitrogen gas through the solution.The reaction mixture was vigorously stirred at that temperature for onehour, then the temperature of the mixture was raised up to 80° C. andthe mixture was again vigorously stirred for one hour. Then 3 g ofsodium carbonate was added to the mixture and the resulting mixture wasstirred at 80° C. for 30 minutes. After addition of 300 ml of a 10%aqueous common salt solution to the mixture and addition of concentratedhydrochloric acid till the pH value of the reaction solution reached 4,the solution was cooled down to 35° C., the precipitates formed werecollected through filtration, followed by washing the precipitates witha 10% aqueous common salt solution and acetonitrile in this order andthen drying to give 68.3 g of the desired intermediate 5 (yield 91% ascalculated on the basis of its disodium salt).

2) Synthesis of Compound 5

The same reaction used in the synthesis of Compound 1 was repeatedexcept that Intermediate 5 was substituted for Intermediate 1 in thefinal step to thus form Compound 5 in a yield of 53%.

EXAMPLE 5 Synthesis of Compound 3

The same reaction used in the synthesis of Compound 1 was repeatedexcept that commercially available N-benzoyl H-acid was substituted forIntermediate 1 in the final step to thus form Compound 3 in a yield of58%.

EXAMPLE 6 Synthesis of Compound 6

1) Synthesis of Intermediate 6 ##STR9##

The intermediate was prepared by an improved Schotten-Baumann method.More specifically, 8-amino-3,6-disulfo-1-naphthol (H-acid, 31.2 g; 91mmol as calculated on the basis of its monosodium salt), sodiumhydroxide (4.5 g, 110 mmol) and sodium carbonate (7 g, 66 mmol) weredissolved in deionized water (200 ml) and a solution of2,5-dichlorobenzoyl chloride (this was prepared by reacting 22 g of2,5-dichlorobenzoic acid with 100 ml of thionyl chloride and thendistilling off the excess thionyl chloride under reduced pressure) inTHF (40 ml) was dropwise added to the resulting solution at 35° to 40°C. for about one hour while blowing nitrogen gas through the solution.The reaction mixture was vigorously stirred at that temperature for onehour, then the temperature of the mixture was raised up to 80° C. andthe mixture was again vigorously stirred for one hour. After addition of200 ml of a 10% aqueous common salt solution to the mixture and additionof concentrated hydrochloric acid till the pH value of the reactionsolution reached 5, the solution was cooled down to 20° C., theprecipitates formed were collected through filtration, followed bywashing the precipitates with a 10% aqueous common salt solution andacetonitrile in this order and then drying to give 48 g of the desiredintermediate 6 (yield 98%, as calculated on the basis of its disodiumsalt).

2) Synthesis of Compound 6

The same reaction used in the synthesis of Compound 1 was repeatedexcept that Intermediate 6 was substituted for Intermediate 1 in thefinal step to thus form Compound 6 in a yield of 63%.

EXAMPLE 7 Synthesis of Compound 7

The route of synthesis of Compound 7 is as follows: ##STR10## 1)Synthesis of Intermediate 7

To a mixed solvent comprising deionized water (20 ml) and concentratedhydrochloric acid (5.1 ml), there was dissolved2,5-diethoxy-4-nitroaniline (4.52 g, 20 mmol) and then a solution ofsodium nitrite (1.56 g, 22 mmol) in deionized water (10 ml) withice-cooling. The reaction mixture was stirred for 60 minutes withice-cooling. The aqueous solution of the diazonium salt thus preparedwas added to a solution of Intermediate 1 (12 g, 22.2 mmol as calculatedon the basis of its disodium salt) and sodium acetate (5.5 g) indeionized water (200 ml) at 10° C. After stirring the reaction mixtureat 20° C. for one hour, the temperature was raised up to 45° C. and themixture was further stirred for additional one hour.

To the reaction mixture, there were added, in order, a 20% sodiumhydroxide solution (9 ml) and sodium sulfide nonahydrate (19.2 g, 80mmol), followed by stirring the mixture at 45° C. for one hour. Aftercompletion of the stirring operation, the reaction mixture wasneutralized by the addition of acetic acid (6 ml) to thus separate outprecipitates. Moreover, a saturated sodium acetate aqueous solution (40ml) was added thereto, followed by collection of the precipitates thusformed through filtration and washing them with a 10% sodium acetateaqueous solution and then isopropyl alcohol.

The crude product of Intermediate 7 thus prepared was suspended in amixed solvent comprising toluene (160 ml) and isopropyl alcohol (40 ml)and then vigorously stirred under reflux. After the completion of thestirring operation, precipitates thus formed were collected throughfiltration, followed by washing them with a mixed solvent of toluene andisopropyl alcohol (volume ratio 4:1) and drying to give 2.8 g ofIntermediate 7 (yield thereof in the form of disodium salt: 19%).

2) Synthesis of Compound 7

A solution of Intermediate 7 (2.23 g, 3.0 mmol) in deionized water (50ml) was ice-cooled, followed by addition of concentrated hydrochloricacid (1.1 ml), vigorous stirring, addition of a solution of sodiumnitrite (230 mg, 3.3 mmol) in deionized water (3 ml) and stirring themixture for 30 minutes with ice-cooling to give a diazonium salt. On theother hand, Intermediate 3 (1.93 g, 3.6 mmol) was dissolved in deionizedwater (40 ml), followed by addition of pyridine (18 ml) and thenaddition of a suspension of the diazonium salt prepared by the foregoingprocedures at a temperature ranging from 10° to 15° C. The reactionmixture was stirred at room temperature for 60 minutes, then heated upto 50° C. and again stirred for additional 30 minutes. Then the reactionmixture was heated to 70° C., followed by addition of isopropyl alcohol(100 ml) and sodium acetate (25 g). After cooling the mixture down toroom temperature, the precipitates thus formed were collected throughfiltration, followed by washing with a 10% aqueous sodium acetatesolution, a mixed solvent of isopropyl alcohol and water (volume ratio4:1) and isopropyl alcohol in this order and drying them.

The crude product of Compound 7 thus prepared was washed with 400 ml ofa hot mixed solvent comprising ethanol and water (volume ratio 4:1),followed by filtration and subsequent washing with ethanol and drying togive 2.46 g (1.91 mmol, yield 64%) of Compound 7.

EXAMPLE 8 Synthesis of Compound 8

The route of synthesis of Compound 8 is as follows: ##STR11## 1)Synthesis of Intermediate 8

There was dissolved p-nitroaniline (4.14 g, 30 mmol) in a mixed solutionof deionized water (30 ml) and concentrated hydrochloric acid (7.5 ml)and a solution of sodium nitrite (2.31 g, 33 mmol) in deionized water(15 ml) was added to the resulting solution with ice-cooling. Thereaction mixture was stirred for 60 minutes with ice-cooling. An aqueoussolution of the diazonium salt thus prepared was added to a solution ofIntermediate 1 (17.7 g, 33 mmol as calculated on the basis of itsdisodium salt) and sodium acetate (9.3 g) in deionized water (270 ml) ata temperature of 10° C. After stirring the reaction mixture at 20° C.for one hour, it was heated up to 45° C., followed by stirring foradditional one hour.

To the reaction mixture, there were added, in order, a 20% sodiumhydroxide solution (15 ml) and sodium sulfide nonahydrate (28.8 g, 120mmol) and the mixture was stirred at 45° C. for one hour. After thecompletion of the stirring, the reaction mixture was neutralized by theaddition of acetic acid (10 ml) to thus separate out precipitates. Afterfurther addition of a saturated sodium acetate aqueous solution (30 ml),the precipitates thus formed were collected through filtration and thenwashed with isopropyl alcohol.

The crude product of Intermediate 8 thus prepared was suspended in amixed solvent comprising toluene (200 ml) and isopropyl alcohol (50 ml)and then vigorously stirred under reflux. After the completion of thestirring, precipitates thus formed were collected through filtration,followed by washing them with a mixed solvent of toluene and isopropylalcohol (volume ratio 4:1) and drying to give 7.3 g of Intermediate 8(yield thereof as calculated on the basis of its disodium salt: 37%).

2) Synthesis of Compound 8

A solution of Intermediate 8 (2.62 g, 4.0 mmol) in deionized water (70ml) was ice-cooled, followed by addition of concentrated hydrochloricacid (1.35 ml), vigorous stirring, addition of a solution of sodiumnitrite (310 mg, 4.4 mmol) in deionized water (10 ml) and stirring themixture for 30 minutes with ice-cooling to give a diazonium salt. On theother hand, Intermediate 3 (2.57 g, 4.8 mmol) was dissolved in deionizedwater (40 ml), followed by addition of pyridine (25 ml) and water (12ml) and then addition of a suspension of the diazonium salt prepared bythe foregoing procedures at a temperature ranging from 10° to 15° C. Thereaction mixture was stirred at room temperature for 60 minutes, thenheated up to 50° C. and again stirred for additional 30 minutes. Thenthe reaction mixture was heated to 70° C., followed by addition ofisopropyl alcohol (100 ml) and sodium acetate (15 g). After cooling themixture down to 40° C., the precipitates thus formed were collectedthrough filtration, followed by washing with a mixed solvent of ethanoland water (4:1), ethanol and isopropyl alcohol in this order and dryingto give 2.46 g of Compound 8 (1.91 mmol, 64%).

EXAMPLE 9 Synthesis of Compound 9

1) Synthesis of Intermediate 9 ##STR12##

The same reaction used for the synthesis of Intermediate 2 in theprocess for synthesizing Compound 1 was carried out except that2-chloro-4-nitroaniline was substituted for 2,5-dimethoxy-4-nitroanilineto thus synthesize Intermediate 9.

2) Synthesis of Compound 9

The same reaction used in the sysnthesis of Compound 8 was carried outexcept that Intermediate 9 was substituted for Intermediate 8 to thusgive Compound 9 in a yield of 23%.

EXAMPLE 10 Synthesis of Compound 10

A solution of 2,5-dimethyl-1,4-phenylenediamine (1.36 g, 10 mmol) indeionized water (100 ml) was ice-cooled, followed by addition ofconcentrated hydrochloric acid (5 ml), vigorous stirring, addition of asolution of sodium nitrite (1.54 g, 22 mmol) in deionized water (10 ml)and stirring the mixture for 60 minutes with ice-cooling to give adiazonium salt. On the other hand, Intermediate 1 (14 g, 26 mmol) wasdissolved in deionized water (120 ml), followed by addition of a 1Nsodium hydroxide solution till the pH of the solution reached about 7,further addition of pyridine (30 ml) and water (15 ml) and then additionof a suspension of the diazonium salt prepared by the foregoingprocedures at a temperature ranging from 10° C. to 15° C. The reactionmixture was stirred at room temperature for 60 minutes, then heated upto 50° C. and again stirred for additional 30 minutes. Then the reactionmixture was heated to 70° C., followed by addition of isopropyl alcohol(200 ml) and sodium acetate (20 g). After cooling the mixture down to35° C., the precipitates thus formed were collected through filtration,followed by washing with a mixed solvent of ethanol and water (volumeratio 4:1) and then ethanol. The crude product of Compound 10 thusprepared was dissolved in 50 ml of water at 80° C. and then ethanol (200ml) was dropwise added to the solution at 80° C. The reaction system wascooled down to 35° C., the precipitates formed were collected throughfiltration, washing with ethanol and drying to give 1.45 g of Compound10 (2.3 mmol, 23%).

EXAMPLE 11 Preparation of Compound 12 by Ion-Exchange Method

Acid-type Amberlite 120B (wet volume 10 ml) was packed in a column,followed by circulation of a large excess of an aqueous solutioncontaining L-tyrosineamide (3.8 g) through the column to thus saturatethe column with L-tyrosineamide through adsorption. Compound 2 (100 mg)was passed, five times (10 minutes/passage), through this ion-exchangecolumn and the resulting eluate was lyophilized to give 120 mg ofCompound 12. The completion of the ion-exchange was confirmed by NMR andelemental analyses.

EXAMPLE 12 Preparation of Compound 11 by Ion-Exchange Method

Compound 11 was prepared by repeating the same procedures used inExample 11, i.e., ion-exchange of Compound 2, except for using anion-exchange column packed with Amberlite 120B which had been saturatedwith potassium ions through adsorption.

The compounds of the present invention prepared by the foregoing methodseach was dissolved in dimethylsulfoxide to a concentration of 1 mg/ml,followed by diluting the solution 100 times with pure water or PBS toprepare a solution for measurement and then each solution was subjectedto determination of maximum wavelength of absorbed light and theabsorbance. The results as listed in the following Table were thusobtained. The unit of λ_(max) is nm. Moreover, the bis-azo compoundsprepared by the method described above in general comprise not less than5% by weight of water through absorption of moisture present in the air,but the foregoing absorbance is not corrected for the presence of water.

                  TABLE 2                                                         ______________________________________                                                   λ.sub.max (Absorbance)                                                              λ.sub.max (Absorbance)                         Compound No.                                                                             (Pure Water) (PBS)                                                 ______________________________________                                        Compound 1 668 (0.388)  668 (0.375)                                           Compound 2 669 (0.469)  668 (0.461)                                           Compound 3 669 (0.510)  623 (0.493)                                           Compound 4 669 (0.505)  613 (0.347)                                           Compound 5 668 (0.448)  667 (0.436)                                           Compound 6 668 (0.369)  668 (0.356)                                           Compound 7 675 (0.484)  671 (0.497)                                           Compound 8 653 (0.661)  604 (0.532)                                           Compound 9 602 (0.356)  603 (0.294)                                           Compound 10                                                                              627 (0.555)  616 (0.469)                                           Compound 11                                                                              670 (0.392)  669 (0.381)                                           Compound 12                                                                              670 (0.357)  670 (0.348)                                           ______________________________________                                    

The compounds listed below can also be prepared according to methodsapproximately identical to those described in Examples 1 to 12.##STR13##

It is known that the azo dyes synthesized from enol type couplingcomponents such as phenol, naphthol or pyrazolone comprise tautomerssuch as azo (enol) type and hydrazo (keto) type isomers, i.e., theyundergo a phenomenon called keto-enol type tautomerism (PHOTOGRAPHICSCIENCE AND ENGINEERING, 1976, 20, P. 155). In the presentspecification, such compounds have been described in the azo (enol) typeforms, but the present invention also includes compounds having hydrazo(keto) tautomeric structures such as those exemplified below. ##STR14##

EXAMPLE 13 Mutagenicity of the Compounds of the Invention

Compounds 1 to 10 of the present invention were inspected for themutagenicity according to the Ames Test. The Ames Test was performedusing the following 6 strains: histidine-requiring Salmonellatyphimurium strains TA 100, TA 1535, TA 98, TA 1537 and TA 1538 andtryptophane-requiring Escherichia coli strain WP2uvr A and carried outby the plate culture method according to the metabolism-activationmethod or without using the metabolism-activation method. As to theevaluation of the results, a compound is estimated to be "positive" ifthe number of reverse mutation colonies is increased to a level of notless than two times that observed for the negative control group andshows dose-dependency, while a compound is evaluated to be "negative" ifthe number of reverse mutation colonies does not reach a level of twotimes the latter.

As a result, all of the compounds 1 to 10 of the present invention werefound to be "negative" in the Ames Test.

COMPARATIVE EXAMPLE 1

Bis-azo compounds such as those listed below as comparative compoundswere also inspected for the mutagenicity by the Ames Test carried outaccording to the procedures disclosed in Example 13. These bis-azocompounds are ones whose N-acyl groups on8-amino-1-hydroxynaphthalene-3,6-disulfonic acid are groups other than2,3-dichlorobenzoyl group, unlike the compounds of the presentinvention. ##STR15##

As a result of various investigations, all of the foregoing comparativecompounds were found to be "positive" in the Ames Test. This clearlyindicates that the bis-azo compounds of the present invention areexcellent and have inventive steps.

It has been reported that the p-phenylenediamine or benzidinederivatives formed through the cleavage of the azo bonds present on theconventional bis-azo compounds by the enzymes present in living bodiesbecome principal causes of the mutagenicity of the compounds and thatthe mutagenicity can be reduced due to effects of substituents on thep-phenylenediamine or benzidine ring, in particular, by the introductionof water-soluble substituents (such as sulfonate residues or carboxylgroups), according to the results of studies on the mutagenicity of theconventional bis-azo compounds. However, the result herein obtainedproves such a new finding that the mutagenicity of bis-azo compounds mayalso be reduced by replacing at least one N-acyl group present on8-amino-1-hydroxynaphthalene-3,6-disulfonic acid, which is quite distantfrom the site regarded as the major cause of the mutagenicity, with2,3-dichlorobenzoyl group.

As has been described above, the bis-azo compounds of the presentinvention are highly safe compounds which are free of such undesiredeffects as mutagenicity. Moreover, the structures thereof are simple andtherefore, they can easily be synthesized and are highly useful as dyesor the like.

We claim:
 1. A bis-azo compound represented by the following formula(1), a tautomer thereof or a salt thereof: ##STR16## wherein Rrepresents a substituted or unsubstituted phenyl group or a substitutedor unsubstituted heteroaryl group and X represents a substituted orunsubstituted phenylene group.
 2. A bis-azo compound represented by thefollowing formula (2), a tautomer thereof or a salt thereof: ##STR17##wherein R represents a substituted or unsubstituted phenyl group or asubstituted or unsubstituted heteroaryl group and A and B may be thesome or different and each represents a member selected from the groupconsisting of a hydrogen atom, an alkyl group having 1 to 4 carbonatoms, an alkoxy group having 1 to 4 carbon atoms and a halogen atom. 3.The bis-azo compound, the tautomer thereof or the salt thereof as setforth in claim 2 wherein A and B are identical to one another and eachrepresents an alkoxy group having 1 to 4 carbon atoms.
 4. The bis-azocompound, the tautomer thereof or the salt thereof as set forth in claim2 wherein A and B are identical to one another and each represents amethoxy group.
 5. The bis-azo compound, the tautomer thereof or the saltthereof as set forth in claim 4 wherein R is a phenyl group substitutedwith a halogen atom.
 6. The bis-azo compound, the tautomer thereof orthe salt thereof as set forth in claim 4 wherein R is a phenyl groupsubstituted with a chlorine atom.
 7. A bis-azo compound selected fromthe group consisting of compounds represented by the following formulas,a tautomer thereof or a salt thereof: ##STR18##
 8. The bis-azo compound,the tautomer thereof or the salt thereof as set forth in claim 4 whereinR is a phenyl group of which at least one o-position is substituted witha chlorine atom.